TheprohormoneconvertasesPC1/3andPC2,encodedbythegenesPCSK1andPCSK2respectively,arethe endoproteolytic enzymes responsible for the liberation of opioid?active peptides from larger precursor proteins.Prohormoneconvertasesplayimportantrolesnotonlyinopioidpeptide?mediatedpainsignaling but also function in many other neuronal circuits, including in reward pathways and in hypothalamic circuits involved in feeding and energy homeostasis. For example, both rare and common variations in PCSK1 function as major risk factors for human obesity, potentially due to deficiencies in hypothalamic peptidergicprocessing.Incollaborationwithclinicianswhohaveidentifiedchildrenwithnovelmutations in PCSK1, we have recently determined that mutant human and mouse PC1/3 proteins are subject to targetingdefectswhicharelikelytoresultinhypothalamicproteostaticstress.Basedonourpriorfinding thatmousePC1/3proteinsoligomerizeduringsynthesis,weproposethatdominant?negativeinteractions play a major role in human PC1/3 heterozygote obesity phenotypes, affecting precursor processing to bioactive peptides involved in satiety signaling. We propose that external stressors will exacerbate even mildformsofPC1/3conformationaldistress,impairingC?terminalcleavageofPC1/3tothesmaller,more active forms. These processes will ultimately converge to strongly impair precursor processing, eg. proopiomelanocortincleavagetobeta?endorphin,ACTH,andmostimportantly,totheanorexicpeptide?? MSH.InthepresentproposalwewilluseCRISPR?engineeredcellmodelstoelucidatethecellbiologyand precursorprocessingefficacyofthreehumanPC1/3variantsandmutantsknowntobestronglyassociated with increased risk of obesity. Secondly, we will create mouse models of two common human PCSK1 obesitymutants,andathirdmodelofararebuthighlyimpairedmutant,toextendfindingsmadeincell culture to actual secretory tissues, and to identify the specific physiologic alterations which underlie the PCSK1?mediated obesity phenotype. Lastly, we will test our hypothesis that processing deficits in proopiomelanocortin?synthesizingneuronsunderlietheobesityphenotypebyselectivelyeliminatingPcsk1 expressioninproopiomelanocortin?expressingcellsusingafloxedPcsk1nullmousemodel.Theresultsof these studies will illuminate the biosynthetic mechanisms controlling hypothalamic peptide production thatcontributetohumansusceptibilitytoavarietyofdiseases,fromobesitytorewardpathwaysindrug addiction.